The goal of this proposal is to understand how the regulatory factors ADP ribosylation factor (ARF), RhoA, protein kinase C (PKC) and phosphatidylinositol bisphosphate (PIP2) interact with phospholipase D (PLD). This study could ultimately lead to the identification of consensus binding sites or domains for these factors on PLD and a variety of other proteins. PLD activity has been found in a number of tissues and cell lines, both normal and transformed. It has been implicated in mitogenesis as well as various diseases such as cancer. A better understanding of the regulation of PLD will help determine the role PLD plays in normal, as well as diseased, states. Two approaches will be taken to accomplish this goal. First, a variety of PLD1 fragments will be used to identify regions on PLD1 that interact with ARF, RhoA, PKC, and PIP2. The fragments will be tested as inhibitors of PLD1 activity. Any fragment that binds a regulatory protein will act as a competitive inhibitor of PLD1 activity. If a truncated protein binds PIP2, it will inhibit PLD1 activity but the inhibition will not be competitive. In addition, filter binding assays will be used to determine direct binding of PLD1 to RhoA, ARF, or PKC. The second approach is to generate a variety of chimeras of PLD1 and PLD2 to identify the regions on PLD1 that confer regulation by ARF, RhoA, and PKC. PLD1, but not PLD2, is regulated by these factors. Therefore, with a variety of chimeras, the ability to regulate the chimeric PLDs with the regulatory proteins should vary. PLD activity assays will be performed on the chimeric forms of PLD. Both of these approaches will be undertaken simultaneously as their results should compliment each other.